Design Of Inhibitors Research Articles

Rational Design of Dual Inhibitors for Alzheimer's Disease: Insights from Computational Screening of BACE1 and GSK-3β.

Alzheimer's disease (AD) is one of the most concerned neurodegenerative disorders across the world characterized by amyloid-beta (Aβ) plaques and neurofibrillary tangles (NFTs), leading to cognitive decline and memory loss. Targeting key pathways involved in AD like Aβ and NFT pathways, are crucial for the development of effective therapeutic strategies. In this study, we aimed to identify and establish promising dual inhibitors targeting BACE1 and GSK-3β, two proteins implicated in Aβ and NFT formation respectively. We have used molecular docking, ADME property analysis, and MMGBSA calculations for the identification of hit molecules and further evaluation of binding affinity, drug-like properties, and stability against BACE1 and GSK-3β. Our results demonstrated strong binding affinities of ZINC000034853956 towards the active sites of both proteins, with favorable interactions involving key residues crucial for inhibitory activity. Additionally, ZINC000034853956 exhibited favorable drug-like properties. MD simulations revealed the stable binding of ZINC000034853956 to both BACE1 and GSK-3β over a 50 ns period, with consistent ligand-protein interactions, such as hydrogen bonding and hydrophobic contacts. These findings highlight the potential of ZINC000034853956 as a promising candidate for AD treatment, acting as a dual inhibitor targeting both BACE1 and GSK-3β. Overall, our study provides valuable insights into the potential of ZINC000034853956 as a dual inhibitor for AD. The strong binding affinity, favorable drug-like properties, and stability observed in MD simulations support its suitability for further optimization and preclinical studies. Further investigations are warranted to elucidate the precise molecular mechanisms and therapeutic benefits of ZINC000034853956. Our findings offer hope for the development of novel therapeutic interventions targeting crucial pathways involved in AD neurodegeneration.

A diarylheptanoid derivative mediates glycogen synthase kinase 3β to promote the porcine muscle satellite cell proliferation: Implications for cultured meat production

Skeletal muscle stem cells, or satellite cells, are vital for cultured meat production, driving proliferation and differentiation to form muscle fibers in vitro. However, these abilities are often compromised after long-term in vitro culturing due to a loss of their stemness characteristics. Therefore, effective pharmacological agents that enhance satellite cell proliferation and maintain stemness ability are needed for optimal cell growth for cultured meat production. In this study, the effects of the identified glycogen synthase kinase 3β (GSK3β) inhibitors, ASPP 049, a diarylheptanoid isolated from Curcuma comosa rhizomes, and CHIR 99021 on porcine muscle satellite cell (PMSC) proliferation and Wnt/β-catenin signaling pathway were investigated. We found that both compounds enhanced cell viability and proliferation while preserving the stemness marker, as evidenced by increased expression of the skeletal muscle stem cell marker, Pax7 protein. Molecular dynamics simulations showed that ASPP 049 and CHIR 99021 exhibited differing binding affinities, primarily through hydrophobic interactions, suggesting potential for the design of more potent inhibitors in the future. Despite its weaker binding, ASPP 049 still showed significant effects on the regulation of the Wnt/β-catenin signaling pathway via increased phosphorylation of GSK3β at Ser9 and decreased the phosphorylation of β-catenin at Ser33, Ser37, and Thr41, thereby subsequently activating Wnt transcriptional activity. This study highlights the potential of ASPP 049 and CHIR 99021 to enhance PMSC proliferation and maintain stemness ability, offering a promising avenue for improving cultured meat production.

Design and research of new virulence factor inhibitors for plant bacterial disease control

In this work, various chalcone derivatives incorporating piperazine-isopropanolamine were elaborately designed and synthesized. The antibacterial efficacy revealed that compound Z6 showed notable biological activity, with a median effective concentration (EC50) of 0.29 mg/L against Xanthomonas oryzae pv. oryzae (Xoo) in vitro, which is around 200 times higher than thiodiazole copper (TC, 91.00 mg/L). Similarly, Z21 exhibited a strong biological response (EC50 = 0.83 mg/L) against Xanthomonas axonopodis pv. citri (Xac), outperforming TC (EC50 = 114.60 mg/L). Results of bioactivity test in vivo demonstrate that Z6 displayed significantly better curative and protective activities (51.5 %, 47.9 %) than TC (47.9 %, 30.5 %) against rice bacterial leaf blight (BLB). Further biochemical studies indicates that Z6 targeted multiple virulence factors and repressed the release of essential nutrients required for bacterial proliferation, thereby disrupting the reduction of cell membrane content of the pathogenic bacteria. According to the research, chalcone derivatives containing piperazine-isopropanolamine have potential to be antibacterial candidates.

Structure-guided inhibitor design targeting CntL provides the first chemical validation of the staphylopine metallophore system in bacterial metal acquisition

To survive in the metal-scarce environment within the host, pathogens synthesize various small molecular metallophores to facilitate the acquisition of transition metals. The cobalt and nickel transporter (Cnt) system synthesizes and transports staphylopine, a nicotianamine-like metallophore, and serves as a primary transition metal uptake system in Gram-positive bacteria including the human pathogen Staphylococcus aureus. In this study, we report the design of the first inhibitor of the Cnt system by targeting the key aminobutanoyltransferase CntL which is involved in the biosynthesis of staphylopine. Through structure-guided fragment linking and optimization, a class of acceptor-adenosine dual-site inhibitors against S. aureus CntL (SaCntL) were designed and synthesized. The most potent inhibitor, compound 9, demonstrated a ΔTm value of 9.4 °C, a Kd value of 0.021 ± 0.004 μM, and an IC50 value of 0.06 μM against SaCntL. The detailed mechanism by which compound 9 inhibits SaCntL has been elucidated through a high-resolution co-crystal structure. Treatment with compound 9 resulted in a moderate downregulation of intracellular concentrations of iron, nickel, and cobalt ions in the S. aureus cells cultured in the metal-scarce medium, providing the first chemical validation of the important role of Cnt system in bacterial metal acquisition. Our findings pave the way for the development of CntL-based antibacterial agents in future.

Elucidating Molecular Mechanism and Chemical Space of Chalcones through Knowledge Graph and Machine Learning: A Combined Bio-Cheminformatic Pipeline

We developed a bio-cheminformatics method, exploring disease inhibition mechanisms using machine learning-enhanced quantitative structure-activity relationship (ML-QSAR) models and knowledge-driven neural networks. ML-QSAR models were developed using molecular fingerprint descriptors and the Random Forest algorithm to explore the chemical spaces of Chalcones inhibitors against diverse disease properties, including antifungal, anti-inflammatory, anticancer, antimicrobial, and antiviral effects. We generated and validated robust machine learning-based bioactivity prediction models (https://github.com/RatulChemoinformatics/QSAR) for the top genes. These models underwent ROC and applicability domain analysis, followed by molecular docking studies to elucidate the molecular mechanisms of the molecules. Through comprehensive neural network analysis, crucial genes such as AKT1, HSP90AA1, SRC, and STAT3 were identified. The PubChem fingerprint-based model revealed key descriptors: PubchemFP521 for AKT1, PubchemFP180 for SRC, PubchemFP633 for HSP90AA1, and PubchemFP145 and PubchemFP338 for STAT3, consistently contributing to bioactivity across targets. Notably, chalcone derivatives demonstrated significant bioactivity against target genes, with compound RA1 displaying a predictive pIC50 value of 5.76 against HSP90AA1 and strong binding affinities across other targets. Compounds RA5 to RA7 also exhibited high binding affinity scores comparable to or exceeding existing drugs. These findings emphasize the importance of knowledge-based neural network-based research for developing effective drugs against diverse disease properties. These interactions warrant further in vitro and in vivo investigations to elucidate their potential in rational drug design. The presented models provide valuable insights for inhibitor design and hold promise for drug development. Future research will prioritize investigating these molecules for mycobacterium tuberculosis, enhancing the comprehension of effectiveness in addressing infectious diseases.

Discovery of Selective PDE1 Inhibitors with Anti-pulmonary Fibrosis Effects by Targeting the Metal Pocket.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with no ideal drugs. Our previous research demonstrated that phosphodiesterase 1 (PDE1) could be a promising target for the treatment of IPF. However, only a few selective PDE1 inhibitors are available, and the mechanism of recognition between inhibitors and the PDE1 protein is not fully understood. This study carried out a step-by-step optimization of a dihydropyrimidine hit Z94555858. By targeting the metal pocket of PDE1, a lead compound 3f was obtained, exhibiting an IC50 value of 11 nM against PDE1, moderate selectivity over other PDEs, and significant anti-fibrotic effects in bleomycin-induced pulmonary fibrosis rats. The structure-activity relationship study aided by molecular docking revealed that forming halogen bonds with water in the metal pocket greatly enhanced the PDE1 inhibition, providing a novel strategy for further rational design of PDE1 inhibitors.

Design and Evaluation of Peptide Inhibitors Targeting the Dimerization of SARS-CoV-2 Main Protease.

The severe acute respiratory syndrome virus 2 (SARS-CoV-2) seriously impacted public health. The evolutionarily conserved viral chymotrypsin-like main protease (Mpro) is an important target for anti-SARS-CoV-2 drug development. Previous studies have shown that the eight N-terminal amino acids (N8) of SARS-CoV Mpro are essential for its dimerization, and are used to design inhibitors against SARS-CoV Mpro dimerization. Here, we established a simple readout assay using SDS-PAGE and Coomassie blue staining to measure inhibitory activity of N8 peptide derived from SARS-CoV-2 Mpro. To optimize its inhibitory effect, we then modified the side-chain length, charge, and hydrophilicity of the N8 peptide, and introduced a mutated Mpro recognition sequence. As a result, we obtained a series of potent peptide inhibitors against SARS-CoV-2 Mpro, with N8-A24 being the most efficient with an IC50 value of 1.44 mM. We observed that N8-A24 reduced Mpro dimerization with an IC50 value of 0.86 mM. Molecular docking revealed that N8-A24 formed hydrogen bond interactions with critical dimeric interface residues, thus inhibiting its dimerization and activity. In conclusion, our study not only discovers a series of peptide inhibitors targeting the SARS-CoV-2 Mpro dimerization, but also provides a promising strategy for the rational design of new inhibitors against COVID-19.

Design and Synthesis of Acyclic Boronic Acid Arginase Inhibitors.

Arginase has long been a target of interest in immuno-oncology, but discovering an orally bioavailable inhibitor is severely constrained by the requisite boronic acid pharmacophore. We began our drug discovery campaign by building off the β-position of the literature inhibitor ABH (1). A divergent synthesis with an Ireland-Claisen rearrangement as the key step allowed access to numerous compounds, some of which we crystallized in the active site of arginase 2. We subsequently used structure-based drug design to further improve the potency of this series, ultimately achieving an inhibitor with an IC50 value of 12 nM. Many compounds in this series were designed to behave as prodrugs, releasing their payload with up to 4-fold improved oral exposure relative to the parent. Subtle stereochemical differences between these various inhibitors and prodrugs had substantial effects on potency and pharmacokinetics.

Design, Synthesis, and Biological Evaluation of New Selective PDE4 Inhibitors for Topical Treatment of Psoriasis.

Psoriasis is a complex and chronic inflammatory disease. Current drugs help control the symptoms of psoriasis but make no cure, urging discovery of novel drugs. We report in this paper the discovery of new phosphodiesterase 4 (PDE4) inhibitors for treatment of psoriasis. We designed and synthesized 45 new compounds, among which 14h exhibited IC50 of 0.57 nM for PDE4D and >4100-fold selectivity over other PDE families. Compound 14h inhibited release of inflammatory cytokines of TNF-α (IC50 = 34.2 μM) and IL-6 (IC50 = 40.9 μM) in Raw264.7 cells and reduced the expression of IL-1β and IL-17A in the skin of psoriasis mice. In addition, 14h alleviated IMQ-induced psoriasis in the mouse model and reduced the erythema level, scales, and thickness of the back skin of the mice. In short, our results suggested that PDE4 inhibitor 14h is a strong candidate for the topical treatment of psoriasis.

Pyrrolo[2,3-D]Pyrimidines as EGFR and VEGFR Kinase Inhibitors: A Comprehensive SAR Review.

Tyrosine kinases are implicated in a wide array of cellular physiological processes, including cell signaling. The discovery of the BCR-ABL tyrosine kinase inhibitor imatinib and its FDA approval in 2001 paved the way for the development of small molecule chemical entities of diverse structural backgrounds as tyrosine kinase inhibitors for the treatment of various ailments. Two of the most prominent tyrosine kinases as drug targets are the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor receptor (VEGFR), as evidenced by the clinical success of their many inhibitors in the drug market. Among several other physiological roles, EGFR regulates epithelial tissue development and homeostasis, while VEGFR regulates tumor-induced angiogenesis. The pyrrolo[2,3-d]pyrimidine nucleus represents a deaza-isostere of adenine, the nitrogenous base of ATP. The recent introduction of many pyrrolo[2,3-d]pyrimidines to the drug market as tyrosine kinase inhibitors makes them a hot topic in the medicinal chemistry research area at the present time. This review article comprehensively sheds light on the structure-activity relationship (SAR) of pyrrolo[2,3-d]pyrimidines as EGFR and VEGFR tyrosine kinase inhibitors, aiming to provide help medicinal chemists in the design of future pyrrolopyrimidine kinase inhibitors.

Discerning potent CSF-1r inhibitors for targeting and therapy of neuroinflammation using computational approaches

Microglia, the primary cellular mediator of neuroinflammation, plays a pivotal role in numerous neurological disorders. Precise and non-invasive quantification of microglia is of paramount importance. Despite various investigations into cell-specific biomarkers for assessing neuroinflammation, many suffer from poor cellular specificity and low signal-to-noise ratios. Colony-stimulating factor-1 receptor (CSF-1R), also known as FMS kinase, has emerged as a promising neuroinflammation biomarker with significant relevance to inflammatory diseases. Additionally, CSF-1R inhibitors (CSF-1Ri) have shown therapeutic potential in central nervous system (CNS) pathological conditions by depleting microglia. Therefore, the development of more specific CSF-1R inhibitors for targeting and treating various CNS insults and neurological disorders is imperative. This study focuses on the search for novel CSF-1R inhibitors. Based on the literature on CSF-1R inhibitors, we proposed and investigated ten ligands as novel CSF-1R inhibitors. Among these, the top three ligands, selected based on their maximum binding scores in docking calculations, are subjected to 100 nanoseconds of molecular dynamics (MD) simulation, alongside three reference ligands. All protein-ligand complexes remain stable throughout the dynamics and exhibit minimal fluctuations during the analysis. The results obtained through this study may prove significant for the future design of CSF-1R inhibitors with potential applications in the field of biomedicine.

Design and Evaluation of Pyridinyl Sulfonyl Piperazine LpxH Inhibitors with Potent Antibiotic Activity Against Enterobacterales

Design and Evaluation of Pyridinyl Sulfonyl Piperazine LpxH Inhibitors with Potent Antibiotic Activity Against Enterobacterales

Structure-based design and synthesis of novel FXIa inhibitors targeting the S2' subsite for enhanced antithrombotic efficacy.

Factor XIa (FXIa), a key component of the intrinsic coagulation pathway, has recently been recognized as a safe and effective target for antithrombotic therapy. Research indicates that FXIa inhibitors can lower bleeding risk compared to novel oral anticoagulants. In this study, we designed and synthesized a series of novel FXIa inhibitors based on the structure of Asundexian, with a particular focus on optimizing the P2' region to enhance binding to the S2' subsite of FXIa. This strategy led to the discovery of compound F47, which demonstrated significantly greater FXIa inhibition (IC50 = 2.0nM) compared to Asundexian (IC50 = 5.0nM). F47 also showed excellent anticoagulant activity in the aPTT assay (EC2x = 0.4μM), with strong efficacy and minimal impact on the extrinsic coagulation pathway. Additionally, F47 exhibited inhibitory activity against plasma kallikrein (PKal), with selectivity comparable to that of Asundexian. The compound also displayed acceptable stability in human liver microsomal stability assays. Molecular modeling revealed that F47 binds tightly to the S1, S1', and S2' pockets of FXIa while maintaining key interactions; notably, its P2' moiety forms two additional π-π stacking interactions with the crucial amino acid TYR143. Further studies demonstrated that F47 exhibits dose-dependent antithrombotic efficacy in a rat FeCl3-induced thrombosis model. Ongoing research aims to further elucidate the potential of compound F47 as a promising lead in antithrombotic therapy.

Advances in utilizing reverse micelles to investigate membrane proteins.

Reverse micelles (RMs) have emerged as useful tools for the study of membrane associated proteins. With a nanoscale water core surrounded by surfactant and solubilized in a non-polar solvent, RMs stand apart as a unique membrane model. While RMs have been utilized as tools to investigate the physical properties of membranes and their associated water, RMs also effectively house membrane associated proteins for a variety of studies. High-resolution protein NMR revealed a need for development of improved RM formulations, which greatly enhanced the use of RMs for aqueous proteins. Protein-optimized RM formulations enabled encapsulation of challenging membrane associated protein types, including lipidated proteins, transmembrane proteins, and peripheral membrane proteins. Improvements in biological accuracy of RMs using phospholipid-based surfactants has advanced their utility as a membrane mimetic even further, better matching the chemistry of the most common cellular membrane lipids. Natural lipid extracts may also be used to construct RMs and house proteins, resulting in a membrane model that better represents the complexity of biological membranes. Recent applications in high-resolution investigations of protein-membrane interactions and inhibitor design of membrane associated proteins have demonstrated the usefulness of these systems in addressing this difficult category of protein. Further developments of RMs as membrane models will enhance the breadth of investigations facilitated by these systems and will enhance their use in biophysical, structural, and drug discovery pursuits of membrane associated proteins. In this review, we present the development of RMs as membrane models and their application to structural and biophysical study of membrane proteins.

Application of the Linear Interaction Energy Method to Nitric Oxide Synthase Structure-Based Inhibitor Design.

The overproduction of nitric oxide by neuronal nitric oxide synthase (nNOS) is associated with several neuropathological conditions. As a result, inhibition of nNOS is a desirable therapeutic goal while avoiding the inhibition of endothelial NOS (eNOS) given its essential role in maintaining cardiovascular tone. Designing inhibitors with high specificity for nNOS over eNOS is challenging given the close similarity in the active site structure of all mammalian NOS isoforms. Computational methods like free energy perturbation (FEP) and thermodynamic integration (TI) offer attractive avenues for rational drug design, but application of these methods to NOS is hindered by several challenges, including proper handling of highly charged inhibitors with diverse structures as well as computational expense. To address these issues, we present a simplified approach combining continuum dielectric generalized born (GB) solvent models with linear interaction energy (LIE) calculations. Our method demonstrates excellent agreement with experimental data for charged inhibitors targeting mammalian NOS isoforms (mNOS). Our results highlight the utility of the GB-LIE method as a promising tool for screening NOS inhibitors and potentially other protein targets with charged active sites and diverse inhibitor structures.

Design and Synthesis of Dual Galectin-3 and EGFR Inhibitors Against Liver Fibrosis.

Liver fibrosis, mainly arising from chronic viral or metabolic liver diseases, is a significant global health concern. There is currently only one FDA-approved drug (Resmetirom) in the market to combat liver fibrosis. Both galectin-3 and epidermal growth factor receptor (EGFR) play important roles in liver fibrosis, while galectin-3 may interact with EGFR. Galectin-3 inhibitors, typically lactose or galactose derivatives may inhibit liver fibrosis. We hypothesized that targeting both galectin-3 and EGFR may have better effect against liver fibrosis. Here, EGFR inhibitor erlotinib was used in a series of designed galectin-3 inhibitors after hybridization with the pharmacophore structure in reported galectin-3 inhibitors to impede hepatic stellate cells (HSCs) activation by a typical method of click chemistry. Bioactivity test results showed that compound 29 suppressed TGF-β-induced upregulation of fibrotic markers (α-SMA, fibronectin-1, and collagen I). The preferred compound 29 displayed better binding to galectin-3 (KD = 52.29 μM) and EGFR protein (KD = 3.31 μM) by SPR assay. Further docking studies were performed to clarify the possible binding mode of compound 29 with galectin-3 and EGFR. Taken together, these results suggested that compound 29 could be a potential dual galectin-3 and EGFR inhibitor as leading compound for anti-liver fibrosis new drug development.

Mixture-of-Experts Based Dissociation Kinetic Model for De Novo Design of HSP90 Inhibitors with Prolonged Residence Time.

The dissociation rate constant (koff) significantly impacts the drug potency and dosing frequency. This work proposes a powerful optimization-based framework for de novo drug design guided by koff. First, a comprehensive database containing 2,773 unique koff values is created. Based on the database, a novel generic dissociation kinetic model is developed with a mixture-of-experts architecture, enabling high-throughput predictions of koff with high accuracy. The developed model is then integrated with an optimization-based mathematical programming approach to design drug candidates with low koff. Finally, the τ-RAMD method is utilized to rigorously verify the designed potential drug candidates. In a case study, the framework successfully identified numerous new potential HSP90 inhibitor candidates, achieving a maximum 45.7% improvement in residence time (τ = 1/koff) compared to that of a known exceptional HSP90 inhibitor. These findings demonstrate the feasibility and effectiveness of the kinetics-guided optimization-based de novo drug design framework in designing drug candidates with prolonged τ.

Screening and design of PARP12 inhibitors from traditional Chinese medicine small molecules using computational modeling and simulation

The poly (ADP-ribose) polymerase (PARP) family of enzymes plays a pivotal role in orchestrating a multitude of cellular processes, including DNA repair mechanisms, transcriptional regulation, and modulation of immune responses. Within this family, PARP12 emerges as a noteworthy candidate for targeted cancer therapeutics. Consequently, this investigation endeavors to screen and design potential PARP12 inhibitors derived from traditional Chinese medicinal compounds by employing sophisticated molecular modeling and computational medicinal chemistry approaches. The compound RBN2397 is utilized as a benchmark, and the binding efficacies of the newly identified small molecules are assessed against a spectrum of criteria, encompassing molecular interactions, binding free energy, and extensive post-simulation analyses. The outcomes demonstrated that the identified small molecules, specifically tcm8650 and its derivative XC-1, possess remarkable binding affinities and exhibit reduced binding free energies compared to RBN2397. The molecular docking and interaction profiles of these compounds were also comprehensively scrutinized. Moreover, ADMET profiling meticulously evaluated the pharmacokinetic profiles and physicochemical characteristics of these promising molecules and their projected human physiological impact. These computational studies indicated their potential therapeutic applicability and predicted acceptable safety profile, advocating their further exploration as viable candidates in cancer treatment.

Structural Studies on Mycobacterial NudC Reveal a Class of Zinc Independent NADH Pyrophosphatase

Non-tuberculous mycobacteria (NTM) have emerged as an increasing threat to public health, due to the extreme antibiotic resistance. NADH pyrophosphatase (NudC) was proposed involving in mycobacterial resistance to the first line anti-tubercular drug isoniazid (INH) or its analog ethionamide (ETH), by hydrolyzing their NAD modified active forms (NAD-INH and NAD-ETH). In this study, we performed enzymatic and structural studies on NudC from M. abscessus (NudCMab), which is highly resistant to isoniazid and emerging as the most worrisome NTM. We determined the crystal structures of NudCMab in apo form, substrate NAD-bound form and product AMP-bound form. We observed the mode for the Nudix motif of NudCMab capturing the pyrophosphate group of NAD mediated by three divalent cation ions, which provides details for understanding the mechanism on NudC hydrolyzing NAD(H) or NAD-capped substrate. Interestingly, our structures revealed a novel subclass NudC from mycobacteria characterized by a unique arginine residue on the conserved QPWPFPxS motif, as well as a unique tower domain that replaces a well-defined zinc-binding motif in E.coli NudC and catalytic domain of mammalian Nudt12. Thus, our structural studies on NudCMab not only present a class of zinc independent NADH pyrophosphatase in mycobacteria, but also may facilitate the design of NudC inhibitors for the treatment of mycobacteria infections in combination with INH or ETH.

Investigating the Inhibition of Diindolylmethane Derivatives on SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (Mpro) is an essential enzyme that promotes viral transcription and replication. Mpro conserved nature in different variants and its nonoverlapping nature with human proteases make it an attractive target for therapeutic intervention against SARS-CoV-2. In this work, the interaction mechanism between Mpro and diindolylmethane derivatives was investigated by molecular docking, enzymatic inhibition assay, UV-vis, fluorescence spectroscopy, and circular dichroism spectroscopy. Results of IC50 values show that 1p (9.87 μM) was the strongest inhibitor for Mpro in this work, which significantly inhibited the activity of Mpro. The binding constant (4.07 × 105 Lmol-1), the quenching constant (5.41 × 105 Lmol-1), and thermodynamic parameters indicated that the quenching mode of 1p was static quenching, and the main driving forces between 1p and Mpro are hydrogen bond and van der Waals force. The influence of molecular structure on the binding is investigated. Chlorine atoms and methoxy groups are favorable for the diindolylmethane derivative inhibitors of Mpro. This work confirms the changes in the microenvironment of Mpro by 1p, and provides clues for the design of potential inhibitors.